Part:BBa_K404246

pCMV_[AAV2]-VP123ex (ViralBrick-587-BAP)
The Biotinylation Acceptor peptide motif, inserted into the 587 loop of pCMV_[AAV2]-VP123

[pCMV_[AAV2-VP123ex(ViralBrick-587-BAP]
BioBrick Nr.	BBa_K404246
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS provided by Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).

CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

Usage and Biology

The Biotinylation Acceptor Peptide (BAP) is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2396
  Illegal XhoI site found at 698
  Illegal XhoI site found at 884
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 665
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2982
  Illegal SapI site found at 1833